Trading of mycotoxin-contaminated agricultural commodities is tightly regulated at both national and international levels. Each year, compliance with these regulations causes the loss of millions of dollars in agricultural produce in the United States alone. Trade sanctions and health effects from mycotoxin-contaminated grains add significantly to the losses (see, e.g., Brown et al. (1996) PNAS USA 93:14873-14877).
Accordingly, it is highly desirable to transform various mycotoxins produced by fungal pathogens in crops into inactive compounds which present no human or animal toxicity. This would alleviate important food pollution problems, as well as lessen the costs associated with complying with detecting and destroying mycotoxin-contamination in various crop commodities. Pioneering work in the of construction of nucleic acids for mycotoxin detoxification was done by co-workers of the inventor, see, WO 00/20573. The present invention extends this work to the detoxification of fumonisins.
The term “mycotoxin” generically refers to a number of toxic molecules produced by fungal species, including polyketides and polyketide derived secondary metabolites (such as fumonisins, aflatoxins, sterigmatocystins, alperisins, trichothecenes, fumifungins, and the like). Polyketides are a large, structurally diverse class of secondary metabolites synthesized by bacteria, fungi, and plants and are formed by a polyketide synthase (PKS) through the sequential condensation of small carboxylic acids. See, e.g., Katz and Donadio (1993) Annu Rev Microbiol 47:875-912; Brown et al. (1996) PNAS USA 93:14873-14877; Silva et al. (1996) J Biol Chem 271:13600-608; Kelkar, I. (1997) J Biol Chem 272:1589-94; Busby & Wogan (1985) in Chemical Carcinogens (Searle ed., 1985) pp. 945-1136, American Chemical Society, Washington D.C.; Kimura et al. (1998) J Biol Chem 273:1654-1661.
Fumonisins are a structurally distinct family of mycotoxins with at least 15 known members, produced by several Fusarium species (see, e.g., Scott (1993) Int J Food Microbiol 18:257-270 and the references therein). Fumonisins have potential toxic and carcinogenic effects in mammals, and have been associated with a number of animal toxicoses, including equine leukoencephalomalacia and porcine pulmonary edema. Fumonisins mimic sphingolipid precursors inhibiting sphingolipid biosynthesis, a property which is thought to be related to their toxic (e.g., hepatotoxic, renotoxic) and carcinogenic effects. For example, Fumonisin B 1 (FB1), the most prevalent of the fumonisins is the diester of propane-1,2,3-tricarboxylic acid and 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyeicosane (empirical formula C34H59NO15).
Fusarium infections are widespread among field grown corn (it should be noted that the terms ‘corn’ and ‘maize’ are used interchangeably herein), and detectable levels of fumonisins, while more prevalent in corn exhibiting signs of physical damage and infestation, can be found worldwide in food and feed products in the absence of overt symptoms, making it difficult to monitor and eradicate this potentially dangerous toxin. Fumonisins are stable upon exposure to light, and can withstand temperatures commonly used during food processing. For example, following dry milling of corn, fumonisins are found in the resulting bran, germ and flour, and are similarly stable in maize and polenta. However, fumonisins can be hydrolyzed upon treatment with hot alkali solution (i.e., as is performed in some grain treatments/preparations).
Biological approaches to detoxifying fumonisins have thus far focused on isolating proteins and nucleic acids from naturally occurring organisms capable of metabolizing fumonisins. For example, esterases capable of degrading fumonisins to their de-esterified form, e.g., amino polyol 1 (AP1) and related compounds have been described (see, e.g., U.S. Pat. No. 5,716,820, U.S. Pat. No. 5,792,931). Similarly, naturally occurring amino polyol amine oxidase (APAO) enzymes capable of oxidatively deaminating AP 1 to the 2-oxo derivative of AP1 or its cyclic ketal form have also been described in WO 00/04159 and WO 00/01460. These naturally occurring APAO enzymes have little activity, however, on intact fumonisins.
The present invention offers new and useful sequences encoding polypeptides with an ability to detoxify fumonisins and fumonisin-derivatives and analogs as well as methods related to detoxification of these mycotoxins. This detoxification is particularly useful in crops, thereby solving each of the problems outlined above, as well as providing a variety of other features which will be apparent upon review.